The long-term objective of this research project is to provide information about the structure-function relationships of voltage-gated potassium (Kv) channels in the heart, and the mechanisms of their modulation by auxiliary subunits and by pharmacology agents. Over the last funding period, we have made progress in the above areas related to 4 Kv channels formed by the following pore-forming subunits: HERG (rapid delayed rectifier, IKr, channel), Kv4.3, Kv4.2 and Kv1.4 (transient outward, Ito, channels). We have also studied the roles of a promiscuous auxiliary subunit, KCNE2, in the function of Ito and IKs (slow delayed rectifier). These results, as well as advances in the ion channel field, help shape the current proposal, which has 3 Specific Aims: (1) Probing the outer vestibule structure of the HERG channel. Our data suggest that the outer vestibule of HERG has a unique structure, that requires data from different approaches before a model can be constructed based on the KcsA coordinates. The first approach is 'peptide toxin footprinting'. We have characterized 2 suitable toxins, and will use one of them, BeKm-1, in the proposed experiments. The second approach is to directly determine the structures of HERG-related peptides in a membrane mimetic environment using the NMR techniques. (2) Probing the packing pattern of alpha-helices in the voltage-sensing domain of the HERG channel. We will use a 'disulfide mapping' approach, or a modified version of it, to identify contact points among S1 - S4 of HERG, and use molecular modeling to construct a model of the voltage-sensing domain. (3) Probing the structural basis for KCNE2 modulation of Kv4.2. We will first test the hypothesis that KCNE2 is intercalated between S5 and S6 of Kv4.2, and then use the disulfide mapping approach to identify the contact points between them. This information will be used in molecular modeling to dock the KCNE2 peptide with the pore domain of Kv4.2. Therefore, we will use techniques of cysteine scanning mutagenesis, mutant cycle analysis, and disulfide mapping to deduce channel surface structure (with help from peptide toxins) and the packing pattern of transmembrane alpha-helices. This information is used in a molecular modeling effort to model the pore domain, the voltage sensing domain and the docking of KCNE peptide with the pore domain. This structural information will be useful for the design and development of therapeutic agents targeting HERG and KCNE.